Name: assessment of calcium sparks in intact skeletal muscle fibers
Uploaded: 2014-02-24T15:00:00
Description: Watch this Scientific Journal Video about Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers at JoVE.com

Dette arbejde blev støttet af RO1-AG028614 til JM, RO1-AR063084to NLW og RO1-AR057404 til JZ.

1.. Opsætning Osmotisk-stress perfusionssystem Figur 1 er et skematisk protokol af calcium gnister vurdering intakte skeletmuskulatur fibre. <ol><li> Opsæt en tre-akse (xyz) mikromanipulator stand til at placere udløb spidsen af ​​perfusionssystemet indeholder mindst to kanaler. Dette kunne gøres med engangs Luer-sprøjtecylinder med vedhæftet tre - vejs Luer - Lok stophane til at tænde og / eller slukke for strømmen af infusionsvæsker. Disse ...

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+amplification+of+ROS+and+Ca2++signals+in+stressed+mdx+dystrophic+skeletal+muscle+fibers.">Reciprocal amplification of ROS and Ca2+ signals in stressed mdx dystrophic skeletal muscle fibers.</a> Pflugers. Arch. 458 (5), 915-928 (2009).</li><li>Lovering, R. M., Michaelson, L., Ward, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malformed+mdx+myofibers+have+normal+cytoskeletal+architecture+yet+altered+EC+coupling+and+stress-induced+Ca2++signaling.">Malformed mdx myofibers have normal cytoskeletal architecture yet altered EC coupling and stress-induced Ca2+ signaling.</a> Am. J. Physiol. Cell. Physiol. 297 (3), 571-580 (2009).</li><li>Weisleder, N., Ma, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++sparks+as+a+plastic+signal+for+skeletal+muscle+health,+aging,+and+dystrophy.">Ca2+ sparks as a plastic signal for skeletal muscle health, aging, and dystrophy.</a> Acta. 27 (7), 791-798 (2006).</li><li>Weisleder, N., Zhou, J., Ma, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+calcium+sparks+in+intact+and+permeabilized+skeletal+muscle+fibers.">Detection of calcium sparks in intact and permeabilized skeletal muscle fibers.</a> Methods Mol. Biol. 798, 395-410 (2012).</li><li>Picht, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SparkMaster:+automated+calcium+spark+analysis+with+ImageJ.">SparkMaster: automated calcium spark analysis with ImageJ.</a> Am. J. Physiol. Cell. Physiol. 293 (3), 1073-1081 (2007).</li><li>Weisleder, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+aging+is+associated+with+compromised+Ca2++spark+signaling+and+segregated+intracellular+Ca2++release.">Muscle aging is associated with compromised Ca2+ spark signaling and segregated intracellular Ca2+ release.</a> J. Cell. Biol. 174 (5), 639-645 (2006).</li><li>Weisleder, N., Ma, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+Ca2++sparks+in+aging+skeletal+and+cardiac+muscle.">Altered Ca2+ sparks in aging skeletal and cardiac muscle.</a> Ageing Res. Rev. 7 (3), 177-188 (2008).</li><li>Yi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+calcium+uptake+regulates+rapid+calcium+transients+in+skeletal+muscle+during+excitation-contraction+(E-C)+coupling.">Mitochondrial calcium uptake regulates rapid calcium transients in skeletal muscle during excitation-contraction (E-C) coupling.</a> J. Biol. Chem. 286 (37), 32436-32443 (2011).</li><li>Tjondrokoesoemo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+1+inositol+(1,4,5)-trisphosphate+receptor+activates+ryanodine+receptor+1+to+mediate+calcium+spark+signaling+in+adult+Mammalian+skeletal+muscle.">Type 1 inositol (1,4,5)-trisphosphate receptor activates ryanodine receptor 1 to mediate calcium spark signaling in adult Mammalian skeletal muscle.</a> J. Biol. Chem. 288, 2103-2109 (2013).</li><li>Martins, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+contribute+to+Ca2++signals+produced+by+osmotic+stress+in+mouse+skeletal+muscle+fibres.">Reactive oxygen species contribute to Ca2+ signals produced by osmotic stress in mouse skeletal muscle fibres.</a> J. 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Denne metode til vurdering af Ca 2 + gnister i intakt skeletmuskulatur er et nyttigt værktøj for muskel fysiologi og sygdom forskning. Vi viste, at Ca2 + gnist reaktion blev ændret i forskellige forhold, herunder muskeldystrofi 11 ældning 18, 19, samt i amyotrofisk lateral sklerose 20. Vores seneste undersøgelse viste også funktionel kobling mellem IP 3-receptoren og RyR repræsenterer en kritisk komponent, der bidrager til Ca 2 +</s...

Intracellulært frit Ca2 + ([Ca 2 +] i) er en alsidig og vigtig sekundær budbringer, der regulerer flere cellulære funktioner i overgearet celler, såsom neuroner, hjerte-, knogle-og glatte muskler (for oversigt se Stutzmann og Mattson 1). Reguleret Ca 2 + mobilisering og cross-talk mellem sarcoplasmic reticulum (SR) og T-tubuli (TT) membraner er grundlæggende regulatorer af muskel fysiologi. Endvidere havde ændringer i Ca2 +-signalering er vist at være en underliggende mekanisme kontraktil dysfunktion i forskellige muskelsygdomme. Ca 2 + gnister er lokaliseret elementære Ca 2 + frigivelse begivenheder stammer fra åbningen af ryanodine receptoren (RyR) kanal på sarcoplasmic reticulum (SR) membran 2. I hjertemusklen opstår gnister spontant gennem åbning af RyR2 kanal ved en Ca2 +-inducerede Ca 2 + release (CICR) mekanisme 3-5. I skeletmuskulatur er RYR1 strengt kontrolleret af spændingssensoren på TT membran 6,7. Således Ca 2 + gnister undertrykkes og sjældent påvist i hvile forhold i intakte skelet muskelfibre. Indtil for nylig, at den sarcolemmal membran behov blive forstyrret af forskellige kemiske eller mekaniske &quot;flåning&quot; metoder til at afkoble undertrykkelsen af spændingssensoren på RYR1 og tilladt for Ca 2 + gnist begivenheder, der skal detekteres i skeletmuskulatur 8,9. En fremgangsmåde beskrevet tidligere krævede permeabilisering af membranen af muskelfibre af saponin vaskemiddel 10. I 2003 opdagede vi, at enten forbigående hypoosmotisk stress eller hyperosmotisk stress kan fremkalde perifer Ca 2 + gnister støder op til sarcolemmal membran i intakte muskelfibre 11. Denne metode er siden blevet modificeret for at studere den molekylære mekanisme og modulering af Ca2 + </sup&gt; frigivelse og dynamik 12-16. Her skitserer vi detaljerne i vores eksperimenterende tilgang til induktion, detektion og analyse af Ca 2 + gnister i intakt skeletmuskulatur. Vi præsenterer også vores specialbyggede gnist analyse program, der kan bruges til at kvantificere de elementære egenskaber af de enkelte Ca 2 + gnister i skeletmuskulaturen, fx gnist frekvens og amplitude (Δ F/F0, hvilket afspejler den åbne sandsynlighed af Ryr kanaler, og Ca 2 + belastning inde i SR), tid til at toppe (stigetid) og varighed (FDHM, fuld varighed på halv maksimal amplitude) af gnister, samt den rumlige fordeling af Ca 2 + gnister. Desuden præsenterer vi beviser for, at links ændret Ca 2 + gnister til de forskellige patofysiologiske tilstande i skeletmuskulaturen, såsom muskelsvind og amyotrofisk lateral sklerose. Fordelen ved denne teknik indebærer evnen til at måle Ca2 + i relativt undamalderen celler, i stedet for stripning muskelfibrene, som muliggør optagelse af Ca 2 + gnister i forhold tættere på fysiologisk. Derudover vores specialdesignede program giver mere nøjagtige beregninger af egenskaber gnisten i forhold til muskelfibre.

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		<col />
		<col />
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		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Dissecting microscope</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td style="width:367px;">Dissect out fibers and check muscle integrity</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Dissection tools -scissors, and fine tip forceps</td>
			<td style="width:111px;">Fine Science Tools</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:195px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td>DOW CORNING</td>
			<td style="width:119px;">184 SIL ELAST KIT</td>
			<td style="width:367px;">&#160;Make ahead of time for fiber dissection. Mix 5 ml liquid Sylgard components and pour into a 60-mm glass Petri dish and waiting 48 hr to let the Sylgard compound become clear, colorless solid substrate.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60-mm glass petri dish&#160;</td>
			<td style="width:111px;">Pyrex</td>
			<td style="width:119px;">3160</td>
			<td>Fiber dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isotonic Tyrode Solution&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Minimal Ca2+ Tyrode Solution</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hypotonic Tyrode Solution&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">collagenase type I&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C-5138</td>
			<td>Fiber digestion&#160;</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Fluo-4 AM&#160;</td>
			<td style="width:111px;">Invitrogen</td>
			<td>F14201</td>
			<td>Fluorescent Ca2+ imaging dyes&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TMRE</td>
			<td style="width:111px;">Invitrogen</td>
			<td>T669</td>
			<td>mitochondrial membrane potential fluorescent dyes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Delta TPG dishes&#160;</td>
			<td>Bioptechs</td>
			<td style="width:119px;">0420041500C</td>
			<td>35 mm glass bottom dish for imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Spark Fit software</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td>data analysis software</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:195px;">Radiance 2100 laser scanning confocal microscope or equivalent microscope</td>
			<td style="width:111px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">3 axis micromanipulator</td>
			<td style="width:111px;">Narishige International</td>
			<td style="width:119px;">MHW-3</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">temperature controllable orbital shaker</td>
			<td style="width:111px;">New Brunswick scientific</td>
			<td style="width:119px;"></td>
			<td>Fiber dissociation</td>
		</tr>
	</tbody>
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assessment of calcium sparks in intact skeletal muscle fibers

Fastholdelse homeostatiske Ca 2 +-signalering er en grundlæggende fysiologisk proces i levende celler. Ca 2 + gnister er de elementære enheder af Ca 2 + signalering i de tværstribede muskelfibre, der vises som meget lokal Ca 2 + frigivelse begivenheder medieret af ryanodine receptor (RyR) Ca 2 + frigive kanaler på sarcoplasmic reticulum (SR) membran. Korrekt vurdering af muskel Ca 2 + gnister kan give oplysninger om de intracellulære Ca 2 + håndtering egenskaber af sunde og syge tværstribede muskler. Selvom Ca 2 + gnister begivenheder er almindeligvis ses i hvile cardiomyocytes, er de sjældent observeret i hvile skelet muskelfibre, der er således et behov for metoder til at generere og analysere gnister i skeletmuskulatur fibre. Detaljeret her er en forsøgsprotokol til måling af Ca 2 + gnister i isolerede fleksor digitorm brevis (FDB) muskelfibre hjælp fluorescent Ca2 + indikatorer og laserscanning konfokal mikroskopi. I denne tilgang er isolerede FDB fibre udsat for forbigående hypoosmotisk stress efterfulgt af en tilbagevenden til isotonisk fysiologisk opløsning. Under disse betingelser, et solidt Ca2 + gnister respons detekteres støder op til sarcolemmal membranen i unge raske FDB muskelfibre. Altered Ca 2 + gnister respons detekteres i dystrofiske eller alderen skelet muskelfibre. Denne fremgangsmåde har for nylig vist, at membran-afgrænset signalering involverer cross-talk mellem inositol (1,4,5)-triphosphat receptor (IP 3 R) og RyR bidrager til Ca 2 + gnist aktivering i skeletmuskulatur. Sammenfattende vores undersøgelser ved hjælp af osmotisk stress, induceret Ca 2 + gnister viste, at denne intracellulære reaktion afspejler en muskel signalering mekanisme i fysiologi og aldring / sygdomstilstande, herunder musemodeller af muskel dystrofi (MDX-mus) eller amyotrofisk lateral sklerose (ALS model).

Tidligere undersøgelser viste, at forbigående hypoosmotisk stress induceret perifer Ca2 + gnister støder op til sarcolemmal membranen i intakte muskelfibre 11. Figur 1 viser billeder af intakte enkelt muskelfibre med glatte sarcolemmal membran karakteristiske, klare og riller. Figur 2 viser typiske Ca2 + gnister (som XY billeder) blev fremkaldt af forbigående (100 sek) behandling med en hypoosmotisk løsning, der svulmer FDB muskelfibre fra ...

Watch this Scientific Journal Video about Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers at JoVE.com

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers (Video) | JoVE

Beskrevet her er en metode til direkte at måle calcium gnister, de elementære enheder af Ca 2 + frigivelse fra sarkoplasmiske reticulum i intakte skeletmuskulatur fibre. Denne metode udnytter osmotisk stress-medieret udløsning af Ca 2 + frigivelse fra ryanodine receptor i isolerede muskelfibre. Dynamik og homeostatiske kapacitet af intracellulær Ca 2 +-signalering, kan anvendes til at vurdere muskelfunktionen hos sundhed og sygdom.

Vurdering af Calcium Sparks i intakte Skeletal muskelfibre

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

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Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

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High-Throughput Contractile Measurements of Hydrogel-Embedded Intact Mouse Muscle Fibers Using an Optics-Based System

High-Throughput Contractile Measurements of Hydrogel-Embedded Intact Mouse Muscle Fibers Using an Optics-Based System (Video) | JoVE

Kontraktile målinger med høj kapacitet af hydrogelindlejrede intakte musemuskelfibre ved hjælp af et optikbaseret system

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches ...

Functional Site-Directed Fluorometry in Native Skeletal Muscle Cells

Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability

Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability (Video) | JoVE 

Funktionel stedstyret fluorometri i indfødte celler til undersøgelse af skeletmuskulaturens excitabilitet

Functional site-directed fluorometry has been the technique of choice to investigate the structure-function relationship of numerous membrane proteins, ...

Quantification of Skeletal Muscle Fatty Infiltration via Decellularization

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration (Video) | JoVE 

Decellulariseringsbaseret kvantificering af skeletmuskulatur fedtinfiltration

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic ...

Construct Design for Transfecting Cysteine-Engineered Voltage-Gated Ca2+ Channels in Murine Isolated Skeletal Muscle Fibers

Construct Design for Transfecting Cysteine-Engineered Voltage-Gated Ca2+ Channels in Murine Isolated Skeletal Muscle Fibers (Video) | JoVE 

Konstruere design til transfektering af cystein-manipulerede spændingsstyrede Ca2+ kanaler i murin isolerede skeletmuskelfibre

Decellularization of Skeletal Muscle

Decellularization of Skeletal Muscle  (Video) | JoVE 

Decellularisering af skeletmuskulatur 